HER2 is overexpressed in 25-30% of breast cancers and is a major contributor to tumor aggressiveness and recurrence. This has led to the development of HER2-targeted therapies such as trastuzumab, which can be effective in patients overexpressing HER2. Accurate assessment of tumor HER2 levels in real time could help personalize HER2-targeted therapy and monitor therapy response. Unlike current methods for assessing HER2 levels such as immunohistochemistry and fluorescence in situ hybridization (FISH), quantitative positron emission tomography (PET) imaging is noninvasive, can be performed repeatedly, and could provide accurate levels of HER2 in real time. Moreover, the high sensitivity of PET imaging could permit the detection of occult micrometastases and possibly HER2-expressing breast cancer stem cells. Nanobodies (Nbs) are single domain antibodies with a tenth of the molecular weight of an IgG, making them an ideal targeting vector for use in combination with the most widely utilized PET radionuclide, 18F. Conventional methods for protein labeling may result in the loss of 18F from tumor cells after a protein undergoes internalization and intracellular catabolism. We plan to extend the residualizing label (RL) approach, used successfully with radioiodine, to synthesize novel reagents designed to generate 18F-labeled catabolites that will be trapped in tumor cells after Nb internalization. Fluorine-18- labeled analogues of N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB), the best residualizing agent that we developed for radioiodination of internalizing biomolecules, will be generated. We seek to combine a new HER2 targeted 2Rs15d Nb developed by our colleague in Brussels, with a novel 18F- labeling chemistry with the goal of generating a PET imaging tool for assessing HER2 levels in breast cancer and thereby for the personalization of HER2 targeted therapy. If successful, the 18F-labeling reagents and methods that we shall develop could be applied to other proteins and peptides targeting HER2 and other internalizing receptors that are over expressed on breast cancers. We propose the following specific aims: 1. Develop prosthetic groups for 18F-labeling of Nbs based on the best Nb radioiodination residualizing agent, N-succinimidyl 4-guanidinomethyl-3-[*I] iodobenzoate ([*I]SGMIB). 2. Label Nbs with these agents and determine the characteristics of the labeled Nb conjugates. 3. Evaluate labeled Nbs in vitro using HER2-expressing breast cancer cells. 4. Evaluate labeled Nbs in vivo in mice bearing breast carcinoma xenografts.